Osteochondral Tissue-On-a-Chip: A Novel Model for Osteoarthritis Research

Abstract

The existing in vitro and in vivo models for studying osteoarthritis have significant limitations in replicating the complexity of joint tissues. This research aims to validate a Tissue-On-a-Chip system for osteoarthritis research. Osteochondral tissues obtained from knee replacement surgeries of patients with osteoarthritis were cultured in an Organ-On-a-Chip system. This system was designed to supply oxygen and glucose to the cartilage from the bone. The distribution of oxygen and glucose was evaluated by fluorescence using Image-iT Green Hypoxia and 2-NBDG, respectively. Cytotoxicity was measured using lactate dehydrogenase (LDH) levels in chip cultures compared to plate cultures (12 tissues per method). Glycosaminoglycans (GAGs), alkaline phosphatase (ALP), Coll2-1, and procollagen type II N-terminal propeptide (PIINP) were measured in the perfused medium of the Tissue-On-a-Chip over a period of 70 days. Fluorescence of Image-iT Green Hypoxia was observed only in the cartilage area, while 2-NBDG was distributed throughout the tissue. An increase in LDH levels was noted in the plate cultures on day 24 and in the Tissue-On-a-Chip cultures on day 63. Compared to the start of the culture, GAG content increased on day 52, while ALP showed variations. A notable increase in GAG, ALP, and Coll2-1 levels was observed on day 59. PIINP levels remained stable throughout the experiment. The validated osteochondral Tissue-On-a-Chip system can replicate the joint microenvironment, with hypoxic conditions in cartilage and normoxic conditions in bone. Tissue survival and component stability were maintained for approximately two months. This platform is a useful tool for evaluating new drugs and represents a viable alternative to animal models.

Keywords: Joint-On-a-Chip; Organ-On-a-Chip; Tissue-On-a-Chip; bone; cartilage; osteoarthritis; osteochondral tissue.

Figure 1

Osteochondral tissues cultured in a Tissue-On-a-Chip device. Hypoxic regions were labeled with Image-iT Green Hypoxia, as shown in (A,B), where fluorescence was observed in the cartilage layer. Bone cells were stained only with DAPI (images captured with 4× and 10× objectives, respectively). Glucose supply was assessed using 2-NBDG; fluorescence was observed in both cartilage and bone, as shown in (C). In (D), a detailed view of the cartilage is presented (images captured with 4× and 10× objectives, respectively).

Figure 2

LDH levels in osteochondral tissue cultured on plates and in Tissue-On-a-Chip. Femur and tibia explants, both lesioned and non-lesioned, from the knees of three patients were used (n = 12 per culture method). A one-way ANOVA test was used to compare each time point with the baseline at day 3: * p < 0.05, ** p < 0.01.

Figure 3

GAG, ALP, Coll2-1, and PIINP content in the perfused medium of the osteochondral Tissue-On-a-Chip determined by ELISA over 70 days. Femur and tibia explants, both lesioned and non-lesioned, from the knees of three patients were used (n = 12). A one-way ANOVA test was used to compare each time point with the baseline at day 3: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All bar graphs are presented as mean ± SEM.

Figure 4

Osteochondral Tissue-On-a-Chip Setup. (A) Diagram of the osteochondral Tissue-On-a-Chip. The Organ-On-a-Chip materials used are gas tight, so oxygen is supplied solely from the culture medium. A syringe perfuses the culture medium at a flow rate of 2 µL/min. The subchondral bone of the osteochondral tissue is placed on the membrane, distributing glucose and oxygen to the cartilage, mimicking physiological conditions. Created with BioRender.com. (B) Organ-On-a-Chip model. The chip is composed of cyclic olefin polymer (COP) and cyclic olefin copolymer (COC). PET inlet and outlet tubes, connected with connectors, facilitate perfusion. (C) Assembly of the osteochondral Tissue-On-a-Chip. The photograph shows multiple chips connected to syringes inside an incubator, with tubes collecting the culture medium. (D) Cutting of osteochondral explants. The explants are sectioned into 3 × 3 × 4 mm pieces using an acrylic cutting block and carbon steel blades.